The present invention is directed to a vector system wherein multiple lentiviral vectors are used to transfer a large number (library) of nucleic acid segments to host cells. Preferably the system uses an inducible expression system to express the nucleic acid segments, and the lentiviral vector are pseudotyped lentiviral vectors. Still more preferably, the system uses nucleic acid segments encoding antibodies that are expressed intracellularly and bind to their target antigens intracellularly (intrabodies).
In recent years considerable effort has been directed at applying gene delivery techniques. That term describes a wide variety of methods using recombinant biotechnology techniques to deliver a variety of different materials to a cell. These methods include, for example, vectors such as viral vectors, liposomes, naked DNA, adjuvant-assisted DNA, gene gun, catheters, etc. The different techniques used depend in part upon the gene being transferred and the purpose therefore. Thus, for example, there are situations where only a short-term expression of the gene is desired in contrast to situations where a longer term, even permanent expression of the gene is desired.
Vectors that have been looked at include both DNA viral vectors and RNA viral vectors. For example, DNA vectors include pox vectors such as orthopox or avipox vectors (see, e.g., U.S. Pat. No. 5,656,465), herpes virus vectors, such as herpes simplex I Virus (HSV) vector [Geller, A. I. et al., J. Neurochem. 64:487 (1995); Lim, F., et al., DNA Cloning: Mammalian Systems, D. Glover, Ed., Oxford Univ. Press, Oxford, England (1995); Geller, A. I. et al., Proc. Natl. Acad. Sci., U.S.A. 90:7603 (1993)]; Adenovirus vectors [Legal Lasalle et al., Sci. 259-988 (1993); Davidson et al., Nat. Genet. 3:219 (1993); Yang et al., J. Virol., 69:2004 (1995)]; and Adeno Associated Virus Vectors [Kaplitt, M. G., et al., Nat. Genet. 8;148 (1994)]. Retroviral vectors include Moloney murine leukemia viruses (MMLV) and human immunodeficiency viruses (HIV) [See, U.S. Pat. No. 5,665,577].
While much attention has been focused on the use of viral vectors, particularly for in vivo therapy, for example, in somatic cell therapy or direct in vivo applications, other applications exist.
For example, a retroviral vector can be used to infect a host cell and have the genetic material integrated into that host cell with high efficiency. One example of such a vector is a modified Moloney murine leukemia virus (MMLV), which has had its packaging sequences deleted to prevent packaging of the entire retroviral genome. However, that retrovirus does not transduce resting cells. Additionally, since many retroviruses typically enter cells via specific receptors, if the specific receptors are not present on a cell or are not present in large enough numbers, the infection is either not possible or is inefficient. Concerns have also been expressed as a result of outbreaks of wild-type viruses from the recombinant MMLV producing cell lines, i.e., reversions.
Recently, attention has focused on lentiviral vectors such as those based upon the primate lentiviruses, e.g., human immunodeficiency viruses (HIV) and simian immunodeficiency virus (SIV). HIV vectors can infect quiescent cells in addition to dividing cells. Moreover, by using a pseudotyped vector (i.e., one where an envelope protein from a different species is used), problems encountered with infecting a wide range of cell types can be overcome by selecting a particular envelope protein based upon the cell you want to infect. Moreover, in view of the complex gene splicing patterns seen in a lentiviruses such as HIV, multivalent vectors (i.e., those expressing multiple genes) having a lentiviral core, such as an HIV core, are expected to be more efficient. Despite the advantages that HIV based vectors offer, there is still a concern with the use of HIV vectors in view of the severity of HIV infection. Thus, means for providing additional attenuated forms that are less likely to revert to a wild type virus are desirable.
Variations can be made where multiple modifications are made, such as deleting nef, rev, vif and vpr genes. One can also have the 3xe2x80x2 and 5xe2x80x2 U3 deleted LTRs.
However, in such instances the vectors are intended to deliver a single heterologous gene or small group of genes.
In recent years, advances such as the use of expression sequence tags (ESTs) have led to the identification of numerous genes, putative genes and their expression products. While comparisons between nucleotide and amino acid sequence may lead to classifications of these genes, putative genes, and expression products, frequently the specific function of the genes product remains unknown. It would be desirable to have a rapid means for identifying the function of such genes and gene products.
Marasco et al. discovered a method by which one could express antibodies within a cell and have them bind to a target within that cell. [See U.S. Pat. No. 5,851,829 to Marasco and Haseltine]. These intracellularly expressed antibodies (intrabodies) can be used in a method of functional genomics. In this manner, one can take a specific unknown gene express its gene product, use that gene product to generate an antibody thereto and use the antibody intracellularly to xe2x80x9cknock-outxe2x80x9d the putative protein in the cell. Thereafter one can compare that cell to a control to determine the effect the loss of its gene product has on the cell in both in vitro and in vivo systems. This method requires generation of a specific antigen and antibody thereto. It would be desirable to have a method to take advantage of the efficiencies of this approach with large numbers of members of a particular group.
In recent years, attention has been directed to developing large libraries consisting of multiple members of related groups. For example, libraries of antibodies, typically monoclonal antibodies. For example, antigen binding antibody fragments have been expressed on the surface of filamentous phage [G. P. Smith, Science 228: 1315 (1985)], and used to create large libraries of such antibodiesxe2x80x94e.g., 107 members or more, referred to as phage display libraries.
In phage display libraries the carboxyl-terminal end of the Fd or Fv region is tethered to a fragment of a phage coat protein, which anchors, for example, Fab fragment to the surface of the phage. The antigen binding site is formed from the combination of the VH and VL-domain. Phage display libraries can be selected for binding to specific antigens by affinity chromatography [R. P. Hawkins et al., J. Mol. Biol., 226: 889 (1992)] or by panning phage on antigen coated surfaces [C. F. Barbas et al., Proc. Natl. Acad. Sci. USA 88: 4363 (1991)]. Antibodies are selected by affinity binding to specific proteins. However, if the antigen has an unknown function, this methodology does not permit you to determine the function of that protein.
It would be highly desirable to have a method where one could look for any molecule resulting in a particular function and rapidly determine that molecule, e.g. protein. It would be very desirable to be able to do this in an automated manner permitting rapid identification of the desired molecule.
We have now discovered a method to identify and obtain a molecule resulting in a desired function from a large pool of molecules. This method involves using a plurality of vectors, wherein the group of vectors contain a plurality of different target molecules. The target molecules can be any molecules having diversity, e.g. genetic diversity. The molecules can be proteins such as antibodies, growth factors, receptors, cytokines, peptides, ribozymes and antisense molecules. Preferably the target molecules are genes encoding proteins such as antibodies. More preferably the nucleic acid sequences are operably linked to an inducible promoter. The vectors can be used to transduce a plurality of cells. Preferably, the vectors contain a marker gene to permit rapid identification and selection of transformed cells. Thereafter, those cells are screened to identify a cell exhibiting a desired phenotype. Cells exhibiting a desired phenotype are selected and the particular target molecule resulting in the phenotype identified.
In one preferred embodiment the plurality of vectors are lentiviral vectors. These lentiviral vectors preferably contain a selectable marker.
The lentivirus vectors include, for example, human immunodeficiency virus (HIV) (e.g. HIV-1 and HIV-2), feline immunodeficiency virus (FIV), or visna virus. A vector containing such a lentivirus core (e.g. gag) can transduce both dividing and non-dividing cells.
The lentiviral virion (particle) is expressed by a vector system encoding the necessary viral proteins to produce a virion (viral particle). Preferably, there is at least one vector containing a nucleic acid sequence encoding the lentiviral pol proteins necessary for reverse transcription and integration, operably linked to a promoter. Preferably, the pol proteins are expressed by multiple vectors. There is also a vector containing a nucleic acid sequence encoding the lentiviral gag proteins necessary for forming a viral capsid operably linked to a promoter. In one embodiment, the gag-pol genes are on the same vector. Preferably, the gag nucleic acid sequence is on a separate vector than at least some of the pol nucleic acid sequence, still more preferably it is on a separate vector from all the pol nucleic acid sequences that encode pol proteins.
In one embodiment, the gag sequence does not express a functional MA protein, i.e. the vector can still transduce cells in the absence of the entire MA or a portion thereof, if a myristylation anchor is provided. This can be accomplished by inactivating the xe2x80x9cgenexe2x80x9d encoding the MA by additions, substitutions or deletions of the MA coding region. Preferably, this is done by deletion. Preferably, at least 25% of the MA coding region is deleted, more preferably, at least 50% is deleted, still more preferably, at least 60%, even more preferably at least 75%, still more preferably, at least 90%, yet more preferably at least 95% and most preferably the entire coding region is deleted. However, in that embodiment, a myristylation anchor (sequence) is still required. Preferably, the myristylation sequence is a heterologous (i.e., non-lentiviral) sequence.
In another embodiment the lentiviral vector is another form of self-inactivating (SIN) vector as a result of a deletion in the 3xe2x80x2 long terminal repeat region (LTR). Preferably, the vector contains a deletion within the viral promoter. The LTR of lentiviruses such as the HIV LTR contains a viral promoter. Although this promoter is relatively inefficient, when transactivated by e.g. tat, the promoter is efficient because tat-mediated transactivation increases the rate of transcription about 100 fold. However, the presence of the viral promoter can interfere with heterologous promoters operably linked to a transgene. To minimize such interference and better regulate the expression of transgenes, the lentiviral promoter is preferably deleted.
Preferably, the vector contains a deletion within the viral promoter. The viral promoter is in the U3 region of the 3xe2x80x2 LTR. A preferred deletion is one that is 120 base pairs between ScaI and PvuI sites, e.g. corresponding to nucleotides 9398-9518 of HIV-1 proviral clone HXB2, encompassing the essential core elements of the HIV-1 LTR promoter (TATA box, SP1 and NF-PB binding sites). After reverse transcription, the deletion is transferred to the 5xe2x80x2 LTR, yielding a vector/provirus that is incapable of synthesizing vector transcripts from the 5xe2x80x2 LTR in the next round of replication. Thus, the vector of the present invention contains no mechanism by which the virus can replicate as it cannot express the viral proteins.
In another embodiment the vector is a tat deleted vector. This can be accomplished by inactivating at least the first exon of tat by known techniques such as deleting it. Alternatively, one can extend the U3 LTR deletion into the R region to remove the TAR element.
Variations can be made where the lentiviral vector has multiple modifications as compared to a wildtype lentivirus. For example, with HIV being nef-, rev-, vpu-, vif- and vpr-. In addition one can have MA- gag, 3xe2x80x2 and 5xe2x80x2 U3 deleted LTR and variations thereof.
The vector(s) do not contain nucleotides from the lentiviral genome that package lentiviral RNA, referred to as the lentiviral packaging sequence. In HIV this region corresponds to the region between the 5xe2x80x2 major splice donor and the gag gene initiation codon (nucleotides 301-319).
The env, gag and pol vector(s) forming the particle preferably do not contain a nucleic acid sequence from the lentiviral genome that expresses an envelope protein. Preferably, a separate vector contains a nucleic acid sequence encoding an envelope protein operably linked to a promoter is used. This env vector also does not contain a lentiviral packaging sequence. In one embodiment the env nucleic acid sequence encodes a lentiviral envelope protein.
In another embodiment the envelope protein is not from the lentivirus, but from a different virus. The resultant particle is referred to as a pseudotyped particle. By appropriate selection of envelopes one can xe2x80x9cinfectxe2x80x9d virtually any cell. Thus, the vector can readily be targeted to a specific cell. For example, one can use an env gene that encodes an envelope protein that targets an endocytic compartment such as that of the influenza virus, VSV-G, alpha viruses (Semliki forest virus, Sindbis virus), arenaviruses (lymphocytic choriomeningitis virus), flaviviruses (tick-borne encephalitis virus, Dengue virus), rhabdoviruses (vesicular stomatitis virus, rabies virus), and orthomyxoviruses (influenza virus).
The preferred lentivirus is a primate lentivirus [U.S. Pat. No. 5,665,577] or a feline immunodeficiency virus (FIV) [Poeschla, E. M., et al., Nat. Medicine 4:354-357 (1998)]. The pol/gag nucleic acid segment(s) and the env nucleic acid segment will when expressed produce an empty lentiviral particle. By making the above-described modifications such as deleting the tat coding region, the MA coding region, or the U3 region of the LTR, the possibility of a reversion to a wild type virus has been reduced.
A desired family of heterologous nucleic acid segments (sometimes referred to as the target molecule) can be inserted into the empty lentiviral particles by use of a plurality of vectors each containing a nucleic acid segment of interest and a lentiviral packaging sequence necessary to package lentiviral RNA into the lentiviral particles (the packaging vector). Preferably, the packaging vector contains a 5xe2x80x2 and 3xe2x80x2 lentiviral LTR with the desired nucleic acid segment inserted between them. The nucleic acid segment can be antisense molecules or more preferably, encodes a protein such as an antibody. The packaging vector preferably contains a selectable marker. These are well known in the art and include genes that change the sensitivity of a cell to a stimulus such as a nutrient, an antibiotic, etc. Genes include those for neo, puro, tk, multiple drug resistance (MDR), etc. Other genes express proteins that can readily be screened for such as green fluorescent protein (GFP), blue fluorescent protein (BFP), luciferase, LacZ, nerve growth factor receptor (NGFR), etc.
When an inducible promoter is used with the target molecule, minimal selection pressure is exerted on the transformed cells for those cells where the target molecule is xe2x80x9csilencedxe2x80x9d. Thus, identification of cells displaying the marker also identifies cells that can express the target molecule. If an inducible promoter is not used, it is preferable to use a xe2x80x9cforced-expressionxe2x80x9d system where the target molecule is linked to the selectable marker by use of an internal ribosome entry site (IRES) [see Marasco et al., PCT/US96/16531].
IRES sequences are known in the art and include those from encephalomycarditis virus (EMCV) [Ghattas, I. R. et al., Mol. Cell Biol., 11: 5848-5849 (1991)]; BiP protein [Macejak and Sarnow, Nature, 353:91 (1991)]; the Antennapedia gene of Drosophila (exons d and e) [Oh et al., Genes and Dev., 6: 1643-1653 (1992)]; those in polio virus [Pelletier and Sonenberg, Nature 334:320325 (1988); see also Mountford and Smith, TIG, 11:179-184 (1985)]. Preferably, the target molecule is operably linked to an inducible promoter. Such systems allow the careful regulation of gene expression. See Miller, N. and Whelan, J., Human Gene Therapy, 8: 803-815 (1997). Such systems include those using the lac repressor from E. coli as a transcription modulator to regulate transcription from lac operator-bearing mammalian cell promoters [Brown, M. et al., Cell, 49:603-612 (1987)] and those using the tetracycline repressor (tetR) [Gossen, M., and Bujard, H., Proc. Natl. Acad. Sci. USA 89:5547-5551 (1992); Yao, F. et al., Human Gene Therapy, 9:1939-1950 (1998); Shockelt, P., et al., Proc. Natl. Acad. Sci. USA, 92:6522-6526 (1995)]. Other systems include FK506 dimer, VP16 or p65 using estradiol, RU486, diphenol murislerone or rapamycin [see Miller and Whelan, supra at FIG. 2]. Inducible systems are available from Invitrogen, Clontech and Ariad. Systems using a repressor with the operon are preferred. Regulation of transgene expression in target cells represents a critical aspect of gene therapy. For example, a lac repressor combined the tetracycline repressor (tetR) with the transcription activator (VP16) can be used to create a tetR-mammalian cell transcription activator fusion protein, tTa (tetR-VP16), with the tetO-bearing minimal promoter derived from the human cytomegalovirus (hCMV) major immediate-early promoter to create a tetR-tet operator system to control gene expression in mammalian cells. Recently Yao and colleagues [F. Yao et al., Human Gene Therapy, supra] demonstrated that the tetracycline repressor (tetR) alone, rather than the tetR-mammalian cell transcription factor fusion derivatives can function as potent trans-modulator to regulate gene expression in mammalian cells when the tetracycline operator is properly positioned downstream for the TATA element of the CMVIE promoter. One particular advantage of this tetracycline inducible switch is that it does not require the use of a tetracycline repressor-mammalian cells transactivator or repressor fusion protein, which in some instances can be toxic to cells [M. Gossen et al., Proc. Natl. Acad. Sci. USA, 89: 5547-5551 (1992); P. Shockett et al., Proc. Natl. Acad. Sci. USA, 92:6522-6526 (1995)], to achieve its regulatable effects. Preferably, the repressor is linked to the target molecule by an IRES sequence. Preferably, the inducible system is a tetR system. More preferably the system has the tetracycline operator downstream of a promoter""s TATA element such as with the CMVIE promoter. See FIG. 4.
The target molecules used preferably have genes encoding antibodies intended to be expressed intracellularly. Antibodies have long been used in biomedical science as in vitro tools for the identification, purification and functional manipulation of target antigens. Antibodies have been exploited in vivo for diagnostic and therapeutic applications as well. Recent advances in antibody engineering have now allowed the gene encoding antibodies to be manipulated so that the antigen binding domain can be expressed intracellularly. The specific and high-affinity binding properties of antibodies, combined with the creation of large human immunoglobulin libraries and their ability to be stably expressed in precise intracellular locations inside mammalian cells, has provided a powerful new family of molecules for gene therapy applications. These intracellular antibodies are termed xe2x80x9cintrabodiesxe2x80x9d [W. Marasco et al., Gene Therapy, 4:11-15 (1997)]. Preferably, the genes encode a single chain antibody. The molecules preferably contain a tag such as HA so the molecule can be identified later.
The antibodies are preferably obtained from a library of antibodies such as a phage display library.
Thereafter the lentiviral vectors are used to transduce a host cell. One can rapidly select the transduced cells by screening for the marker. Thereafter, one can take the transduced cells and grow them under the appropriate conditions or insert those cells e.g. spleen cells or germ cells, into a host animal.
The promoter is induced and then one screens for cells and/or animals displaying a particular phenotype. Using the tag contained on the molecule, e.g. antibody, one can obtain the molecules, e.g., antibody that resulted in the desired phenotype. In one example, the antibody can then be used identify the antigen it bound to, if that is desired.
This method permits one to use a multitude of molecules to identify a specific molecule providing the desired function from a large group of molecules without first needing to know the specific identity of any member.